Abstract:
During the Avian Influenza (AI) outbreaks in different areas of Pakistan (2003 - 06), a
number of Avian Influenza Virus (AIV) isolates were recovered from the clinical samples.
The samples were subjected to comparative diagnostic evaluation using in-ovo
propagation, Virus Neutralization Test (VNT), rapid detection kits and Reverse
Transcriptase- Polymerase Chain Reaction (RT-PCR). The data revealed that RT-PCR
technique was most sensitive and specific for the detection of Avian Influenza Virus
subtypes and for differentially diagnosing it from other avian respiratory pathogenic
viruses. These isolates were further utilized for the development of multiplex RT-PCR. A
multiplex reverse transcriptase polymerase chain reaction (mRT-PCR) was developed
and standardized for the detection of type A influenza viruses, Avian Influenza Virus
(AIV) subtype H7, H9 and H5 haemagglutinin gene with simultaneous detection of 3
other poultry respiratory pathogens Newcastle disease virus (NDV), infectious bronchitis
virus (IBV) and infectious laryngotracheitis virus (ILTV). Seven sets of specific
oligonucleotide primers were used in this study for the M-Gene of AIV and
haemagglutinin gene of subtypes of H7, H9 and H5 of AIV. Three sets of other specific
oligonucleotide primers were used for the detection of avian respiratory pathogens other
than AIV. The mRT-PCR DNA products were visualized by Agarose Gel Electrophoresis
and consisted of DNA fragments of 1023bp for M-Gene of AIV, 149bp for IBV, 320bp
for NDV and 647bp for ILTV. The second set of primers used for m-RT-PCR of H7N3,
H9N2 and H5N1 provided DNA products of 300bp for H7, 456bp for H5 and 808bp for
H9. The mRT-PCR products for the third format consisted of DNA fragments of 149bp
for IBV, 320bp for NDV, 647bp for ILTV, 300bp for H7, 456bp for H5, 808bp for H9.
The sensitivity and specificity of mRT-PCR was determined and the test was found to be
sensitive and specific for the detection of AIV and other poultry respiratory pathogens. In
the present study, multiplex PCR technique has been developed to simultaneously detect
and differentiate three most important subtypes of AIV’s alongwith 3 most common
avian respiratory pathogens prevalent in poultry in Pakistan.
The non-structural 1 (NS1) protein of avian influenza viruses has been earlier described
as a remarkably conserved protein amongst type A influenza viruses, however with
xxivsubsequent findings of is truncation during extensive circulation in poultry has led to
further investigate its mutation in association with point mutations simultaneously
occurring in more variable genes such as HA and NA. Apart from affecting any of the
biological functions of these viruses, these mutations may affect the immunogenic
component(s) of these viruses, affecting the efficacy of prevalent vaccines. To establish if
Pakistani H7N3 Avian influenza viruses undergo any truncation in non-structural genes,
the non-structural gene 1 (NS1) of 22 H7N3 Avian influenza A viruses isolated from
commercial and domestic poultry was sequenced and compared phylogenetically. The
isolates included in the present study were both of low pathogenecity (LPAI) and highly
pathogenic strains (HPAI) of H7N3 avian influenza viruses as observed in the field with
regards to their mortality rates. These isolates circulated in N.W.F.P, Punjab, and Sindh
areas of Pakistan from 1995 to 2005. Size variation in the predicted amino acid sequence
of each NS1 was revealed with two different levels of carboxy-terminal truncation in
those isolates. Of the 22 isolates analyzed, 02 isolates A/Chicken/Pakistan/NARC-100/04
and A/Chicken/Pakistan/NARC-1282/04 encoded a full length NS1 protein of 230 amino
acids, whereas 20 encoded a truncated protein of 217 amino acids. The isolates exhibiting
the truncated carboxy terminal NS1 protein, clustered together and appeared to be closest
to A/Duck/Jiang Xi/6146/03 (H5N3), A/Duck/Hong Kong/610/79 (H9N2) and A/Aquatic
Bird/Korea/CN-1/04 (H3N6) at the nucleotide level and amino acid level. In contrast, the
nucleotide sequence of one of the isolates with the full length NS1 protein
(A/Chicken/Pakistan/NARC-1282/04) showed 99.9% nucleotide homology and 99.6%
homology to a set of Italian H7N3 isolates of Turkey from 2002 at the NS1 gene e.g
A/turkey/Italy/8912/2002(H7N3) and A/turkey/Italy/214845/02(H7N3). The other isolate
(A/Chicken/Pakistan/NARC-100/04) with the full length NS1 protein showed the highest
homology
(96%)
with
the
NS1
gene
of
an
H5N7
subtype
virus
A/mallard/Denmark/64650/03.
Out of these 22 H7N3 isolates sequenced for the NS1 gene, 6 isolates from the Northern
Parts of Pakistan were further sequenced for the HA and NA genes. One of the isolates
had an untruncated NS1 whereas 5 were truncated. The 5 H7N3 isolates with truncated
NS1 sequenced were HPAI, for the HA gene and showed the presence of typical highly
pathogenic pattern of deduced amino acid sequence at the HA cleavage site. The
xxvphylogenetic analysis of these H7N3 isolates indicated a close resemblance to other
Pakistani isolate sequences in the GenBank, with the next closest resemblance to the
H7N3 isolate from a Peregrine Falcon in U.A.E in the GenBank besides the other
Pakistani isolates. The untruncated isolate for the NS1 gene, A/Chicken/Pakistan/NARC-
1282/04, showed a typical low pathogenicity cleavage site sequence at the HA cleavage
site. Phylogenetic Analysis of this isolate indicated a close resemblance to Italian H7N3
isolates especially A/Chicken/Italy/682/2003 (H7N3) and A/turkey/Italy/8535/2002
(H7N3). The NA gene was analyzed for the presence or absence of a stalk region in the
isolates sequenced. The 5 truncated H7N3 isolates for the NS1 Gene and HP for HA gene
had a stalked NA protein as in H7N3 isolates reported in wild birds showing a close
resemblance to other previously sequenced H7N3 Pakistani isolate sequences in the
GenBank, whereas the untruncated NS1 H7N3 isolate also showing a LPAI cleavage site
sequence A/Chicken/Pakistan/NARC-1282/04 had a deleted NA stalk region, deduced
amino acid sequence showing a deletion of 24 amino acids in concordance with other
Italian H7N3 isolates reflecting a probable introduction of a highly circulating virus in
domestic poultry. It was concluded from the present study that the H7N3 isolates from
Pakistan show slow antigenic drift and continue to evolve in a slow manner during a ten
year period in the poultry population. With information obtained from the data on NS1,
HA1 and NA, continuous monitoring of circulating viruses is possible and subsequent
production of homologous vaccines from field strains is key to the control of HPAI in
poultry.